Process and product for inhibiting or preventing bacterial infections

ABSTRACT

The combination of a polymyxin composition and a hop composition produces unexpected and synergistic activity against gram positive bacteria.

FIELD OF THE INVENTION

The present invention generally relates to processes and products for inhibiting bacteria which cause infections in animals, including human beings, either by treating subjects having a bacterial infection, or treating those susceptible to such infections. More specifically, it concerns processes and products for treating infected or susceptible animals with a mixture of a hop composition and a polymyxin composition.

BACKGROUND OF THE INVENTION

The two compositions, a mixture of which constitute the present invention, are well known individually. Thus, polymyxin compositions, most notably polymyxin B, are recognized anti-bacterial compounds. Polymyxin B is a cyclic polypeptide produced and secreted by Bacillus polymyxa. The anti-bacterial effect of this antibiotic is apparently based on its ability to increase the permeability of the cell wall of bacteria. This creates channels in the cell membrane, resulting in the leakage of small molecules, such as phosphates, out of the bacterial cell. However, polymyxin B's spectrum of activity is recognized as being limited to gram-negative rods, such as Pseudomonas, E. coli, Enterobacter, Klebsiella, Salmonella and Shigella species. The Proteus group is resistant, as are gram-negative cocci and all gram-positive bacteria, in common with yeast and fungi. Apparently the thickness of the cell wall of bacteria affects the ability of polymyxin to permeate the wall and reach the cell membrane.

Polymyxins are produced by the growth of Bacillus polymyxa. A plurality of isomers are found to be present, and thus the family of polymyxins is presently known to consist of polymyxin A, B, C, D and E, the last named also being known by the by the name, colistin. However, polymyxin B is most commonly used. It is quite water soluble and is stable such that a solution of polymyxin B can be maintained under refrigeration for one year without the loss of substantial anti-bacterial activity. While the individual experiments on which the present invention is based have taken place using polymyxin B, which has the greatest anti-bacterial activity of the polymyxins, when the term, polymyxin composition is used herein, it is intended that it cover not only polymyxin B, but other polymyxins such as polymyxins A and E.

The other ingredient that forms the basis of my anti-bacterial composition is a hops composition. The hops plant, Humulus lupulus, is a twining vine that is a member of the mulberry family (Cannabinaceae). The female hop plant has inconspicuous flowers, and forms glandular, cone-shaped catkins which, when ripe and dried, find familiar use to impart a bitter flavor to beverages derived from malt. Hop has been used to flavor and preserve wort and beer since the 12th century in Germany and the 15th century in England. Its resins, which reside in the yellow glands of its cones, are known to possess antimicrobial properties. Those resins are extracted from the cones, usually by supercritical carbon dioxide treatment or by extraction with organic solvents. They are today frequently added for the bittering of beer in place of the hop flowers and hop pellets previously utilized.

With respect to the chemical nature of hop compounds, these are weak acids classified as alpha and beta resins or bitter acids. Alpha bitter acids are represented by humulone and its cogeners, cohumulone, adhumulone, prehumulone and posthumulone. The beta resins are represented by lupulone, often referred to as lupulon, and its cogeners, colupulone, adlupulone, and prelupulone. The alpha and beta acids have alicyclic structures (2,4-cyclohexodine-1-one), but their cogeners differ in the nature of the acyl side chains. While the alpha acids and their derivatives, the iso acids, contribute most to the bitterness of beer, there are many other, sometimes minor constituents that contribute to preservation and bitterness. One of these compounds is xanthohumol, a compound that falls within the scope of the present invention.

The antimicrobial properties of hop compounds are well-known; their extracts having been used to inhibit the growth of gram positive microbes and they are usually inactive against gram negative microbes. Yeast is not inhibited by hop compounds. Fungi are either not inhibited or inhibited only at unfavorably high concentrations of hop extracts. As the term, hop composition, is used herein, it is meant to cover the primary hop compounds, humulone, lupulone as well as xanthohumol, and their cogeners and isomers, either in pure form or as extracts, as well as iso and other derivatives.

SUMMARY OF THE INVENTION

Whether in the form of a process for preventing or inhibiting the growth of bacteria or as a composition that is useful in inhibiting bacterial growth, the present invention is based on the unexpected finding that there is a synergism between polymyxin and hop compositions in inhibiting bacteria that greatly exceeds their individual anti-bacterial capacities. While the reason for such cooperation is not known, the results of the antibacterial activity of the resultant mixture of polymyxin/hop compositions are apparent, surprising and nonobvious.

In its process form, my invention may be broadly defined as a process for inhibiting the growth of bacteria in an animal, which term includes the genus homo sapiens, by treatment with an antibacterial composition, which comprises administering to the animal a pharmacologically effective amount of a mixture of a polymyxin composition and a hop composition, whereby the mixture has synergistic effect on the infection that is greater than the antibacterial effect of the polymyxin composition or the hop composition separately administered. The bacteria toward which the ultimate composition or mixture is directed are usually gram positive.

More specifically defining the process of my invention, the polymyxin compound that will preferentially be utilized is polymyxin B. Even more specifically, the preferred polymyxin is polymyxin B sulphate.

With respect to the hop compounds that find ready use in the hop/polymyxin composition, the β form of the hop composition, also known as lupulone or β acid or β resin, has been found to be preferable in use, at least as presently advised, although the α resin, humulone, as well as iso derivatives, are also suitable for use, as is xanthohumol. Exemplarily, the active ingredient in the hop composition may be isohumulone or tetrahydroisohumulone.

In the product form of the invention, the mixture of compositions that is utilized to treat the bacterial infection in the process form of the invention is separately identified. Here, again, polymyxin B is the presently preferred form of the polymyxin composition, and the α and β acids or resins and xanthohumol may be advantageously utilized as the hop composition that forms part of the mixture of compositions. The physical form of the mixture is related to the use to which the mixture is to be put. Thus, where the mixture is to be used as an eye drop, the mixture will usually take the form of a flowable liquid. However, where the mixture is to be applied to a wound to prevent bacterial infection, it may be preferable that it be in the form of a gel, cream or ointment.

As a consequence, it is a primary object of my invention to provide an effective treatment either for a bacterial infection already present, or as a protective means of warding off such an infection in a situation where there is a possibility that such infection will occur. These and other objects, features and advantages of the invention will be more apparent from a description of a preferred embodiment of the invention and its attendant efficacy, as disclosed hereinafter.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Various hop compositions, including the α and β resins, humulone and lupulone, and xanthohumol, were obtained from S. S. Steiner, Inc., of New York, N.Y. Quantities of polymyxin B were obtained from other commercial sources. In order to test the synergistic effect of mixtures of hops compositions and polymyxin B, two standard tests were used: the paper disc diffusion method, and the minimum inhibitory concentration (MIC) method.

The first test method to be applied was the paper disc diffusion susceptibility method. In this method a filter paper disc impregnated with a chemical is placed on agar, and the chemical will diffuse from the disc into the agar. If an organism is placed on the agar, it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as the zone of inhibition. This disc diffusion method for antibiotic susceptibility is known as the Kirby-Bauer method. In testing the effectiveness of various antibiotics, all factors, e.g., the agar utilized, the depth of the agar, the amount of organisms used, and incubation conditions, are maintained constant for the various organisms and antibiotics being tested. Under these conditions, the zone of inhibition will be the only variable, and the greater the zone, the more effective the antibiotic. The size of the zone of inhibition of the particular antimicrobial substance may also be influenced by the diffusion capability of that substance.

In performing the disc diffusion method tests, a bacterial suspension having an inoculum density of approximately 1×10⁸ CFU/ml (colony forming units) was added to 10 ml trypticase soy agar at 46° C. It was mixed thoroughly by vortexing and then poured into a plastic Petri dish. After hardening of the agar, filter paper discs (φ7 mm.) made from Whatman filter paper that had been impregnated by the manufacturer, Becton Dickinson, Inc., with the suitable antibiotic, were placed on the agar. In this series of tests the ability of each antibiotic per se to control the growth of the bacteria was measured.

Another series of agar containing Petri dishes was then prepared using the same bacteria and density thereof as had been prepared for the prior test series. However, here hop compounds were also present in the agar. The hop extract, either lupulone, humulone or xanthohumol, was dissolved in ethanol. While certain of the hop components are only slightly soluble in water, they are very soluble in ethanol. An ethanol control was prepared in the same way. The various hop fraction solutions were diluted in such a way that when added to the media solutions, the 10 ml. of agar poured into the Petri dish would contain in addition to the hop compound, exactly 1% of ethanol, which in a control was found to be non-inhibitory to the bacteria.

In each test, after an incubation period of 24 hours, a homogeneous lawn of bacteria developed around the paper discs, and a clear zone of inhibition was evident around those discs that contained inhibitory material. The size of the inhibition zones was measured to the nearest mm. The test results are demonstrated in the Tables below. Table 1 evidences the results of disc diffusion assays where the bacterium was Staphlococcus saprophyticus, a gram positive bacterium. As will be evident from the results of Table 1, the control, which was the antibiotic per se, had no effect whatsoever on the bacterium, that is, there was no inhibition zone whatsoever around the disc of filter paper when polymyxin B was used alone. However, when the same polymyxin B was used in conjunction with 0.2 μg/ml of β-resin, the result was a zone of inhibition of 9 mm. This increase was unexpected and unanticipated. The non-obviousness of the increase in the zone of inhibition when a combination of polymyxin and hops are used is made evident by the same test when performed using the same hop compound in conjunction with other antibiotics. The results were uniformly negative, i.e., there was no significant increase in the zone of inhibition when the resin lupulone was added in the same concentration in which it was added to the polymyxin composition. As the results of experiment 1 in Table 1 show, neomycin per se had a zone of inhibition of 23 mm. When the lupulone was added, the zone increased to 25 mm. When the hop compound was added to streptomycin and erythromycin, the zones of inhibition remained constant, as they did with bacitracin. With penicillin, chloramphenicol and tetracycline, there was a decline in the zone.

The experiment was performed again and the results are seen in experiment 2. It will there be seen that with all of the antibiotics other than polymyxin B, there was no significant change in the radius of the zone of inhibition when the β-resin was present. However, when the hop compound was added to the polymyxin B compound, the increase in the zone was dramatic: from zero to 8 mm.

TABLE 1 Experiment 1 Experiment 2 0.2 μg/ml 0.2 μg/ml ANTIBIOTIC CL* β-RESIN CL* β-RESIN POLYMYXIN 0 9 0 8 NEOMYCIN 23 25 26 24 STREPTOMYCIN 15 15 15 14 ERYTHROMYCIN 23 25 23 23 PENICILLIN 41 39 37 35 BACITRACIN 0 0 0 0 CHLORAMPHENICOL 29 27 25 25 TETRACYCLINE 25 15 14 14 CL*—Control

TABLE 2 E.COLI - (Disc diffusion assay in mm.) Experiment 1 Experiment 2 Experiment 3 ANTIBIOTIC CL* 50 μg/ml β RESIN 100 μg/ml β RESIN CL* 10 μg/ml β RESIN 50 μg/ml β RESIN CL* 10 μg/ml β RESIN 50 POLYMYXIN 18/19 17 17/18 18 18 18 18 18 18 NEOMYCIN 17 15 15 16 16 15 16 16 16 STEPTOMYCIN 15 14 13 14 14 13 14 15 14 ERYTHROMYCIN 0 0 0 0 0 0 0 0 0 PENICILLIN 0 0 0 0 0 0 0 0 0 BACITRACIN 0 0 0 0 0 0 0 0 0 CHLORAMPHENICOL 12 12 12 13 16 26 11 11 13 TETRACYCLINE 14 14 14 14 21 15 14 14 14 CL* - Control

For the purpose of showing the lack of response, or synergism, when a gram negative bacterium is treated, Table 2 tabulates the response of various antibiotics and a hop compound when used with E. coli. As is apparent from this table the beta hop compound, when added at the rate of 10 μg/ml, had no effect whatsoever on the ability of the polymyxin or any other antibiotic to inhibit E. coli bacteria. The same was true when the concentration of the hop compound was increased 5-fold to 50 μg/ml.

Another method of determining antimicrobial activity is to assess the minimum inhibitory concentration (MIC) of the composition under test. The MIC is defined as the lowest concentration of the test compound where the bacterium shows no visible growth (Carson et al. 1995). This technique shows that at certain concentrations a test compound has no effect on the growth of a bacterium relative to the control. Thereafter, increased concentrations will have a limiting effect on growth, until the concentration is reached where no growth relative to the control is observed. That point of no growth is the minimum inhibitory concentration.

The MIC was determined by inoculating a bacterial suspension into 10 ml of Trypticase soy broth so as to provide an initial population of 1×10⁵ CFU/ml with different concentrations of hop compounds ranging from 0.1 μg/ml to 10 μg/ml. A positive control with the bacterium alone was prepared as well as an ethanol control. The tubes were incubated at 37° C. for 24 hours and growth of the bacterium was determined by observing the change in turbidity. The results are tabulated in Table 3 below, where, as indicated in the legend, the letter P stands for polymyxin B sulfate, the α and β symbols stand for the corresponding hop resins or bitter acids, and X stands for xanthohumol.

TABLE 3 Activity of antimicrobial agents on various microbes by combinations of hop constituents with polymyxin B sulfate: P α β X α + P B + P X + P exp 1 B.subtilis >20 <30 >3 <10 >1 <3 >1 3 + 20 no syn 1 + 10 exp 2 B.subtilis >20 <30 >3 <10 >1 <3 >1 1 + 10 0.3 + 10 1 + 10 exp 1 B.megaterium >100 >5 <10 >3 10 >3 + 100 3 + 100 3 + 100 exp 2 B.megaterium >100 >5 <10 3 10 >3 + 100 no syn 3 + 100 exp 1 S.salivarius >100 >30 >5 <10 >10 30 + 100 no syn 10 + 100 exp 2 S.salivarius >100 >30 >5 <10 >10 10 + 100 3 + 100 10 + 100 exp 1 S.saprophyticus >100 >10 <30 >1 <3 >1 <10 3 + 30 1 + 30 1 + 30 exp 2 S.saprophyticus >100 >10 <30 >1 <3 >1 <10 3 + 30 1 + 30 1 + 30 P = polymyxin B sulfate α = alpha resin (or humulone) β = beta resin (or lupulone) X = xanthohumol μg = micrograms All numerical values are in pg/ml and show minimum inhibitory concentration.

To further explain the results of Table 3, utilizing B. subtilis as the bacterium, when polymyxin alone was utilized as the inhibitory agent, inhibition was achieved with more than (>) 20 micrograms per milliliter but less than (<) 30 micrograms per milliliter concentration of solution. Following across the Table, and with regard to experiment 1, it required more than 3 but less than 20 μg per ml. to achieve minimum inhibition when the cc resin was used alone, and more than 1 but less than 3 μg when the β resin was utilized. When xanthohumol alone was the inhibitory agent, more than 1 μg was required. With the addition of a hop resin to polymyxin B, the results for this same bacterium in the first series of experiments showed that complete inhibition was achieved using 3 parts of α resin and 20 parts of polymyxin. Inhibition was also achieved using 1 part of xanthohumol and 10 parts of polymyxin. When the B. subtilis test was repeated in experiment 2, the results were the same for the hop resins and polymyxin alone. However, inhibition was achieved with only 1 μg of alpha resin plus 10 μg per ml of polymyxin, and even less, 0.3 μg/ml β resins with 10 μg/ml polymyxin.

Each experiment for a specific bacterium was repeated once, so that there are two experiments for each bacterium. In a small minority of experiments no synergism was apparent, and these instances are duly reported. Yet each time that no synergism was reported, a complementary experiment on the same gram positive bacterium evidenced the presence of synergism. Thus, it is concluded that the experiments where no synergism was shown, yielded that result because of the experimental error. The overall conclusion, however, from the results of Table 3, is that when a hop compound is used in conjunction with polymyxin B, the amount of that anti-bacterial is greatly reduced and, therefore, that when various hop compounds are combined with polymyxin, the effect of such combination is to greatly reduce the quantity of polymyxin necessary to inhibit the action of the bacteria that were the subjects of the experiments.

It will thus be apparent that the combination of a hop compound, either the alpha or beta resins or xanthohumol, or combinations thereof, have a synergistic effect on polymyxin when the two compositions are used in combination. For reasons not determined, this synergistic effect is not apparent when hop compounds are used in combination with other antibiotics, at least with such other antibiotics as have been tested. The synergistic effect results in a far more potent antibiotic preparation, and one that may well eliminate the need for combinations of antibiotics now on the market, such as a combination of polymyxin and bacitracin.

It will also be recognized by those of skill in this art that certain modifications and alterations in the preferred embodiments of my invention will be obvious without departing from the spirit of the invention. As to all such obvious modifications are desired to be included within the purview of my invention, which is to be limited only by the scope, including equivalents, of the following, appended claims. 

1. A process for inhibiting bacterial infections in animals, comprising administering to said animals a pharmacologically effective amount of a mixture of a polymyxin composition and a hop composition, said mixture having a synergistic effect on said infection greater than the antibacterial effect of said polymyxin composition and said hop composition separately administered.
 2. A process as claimed in claim 1, in which said infection is caused by gram positive bacteria.
 3. A process as claimed in claim 1, in which the active ingredient of said polymyxin composition is polymyxin B.
 4. A process as claimed in claim 1, in which an active ingredient of said hop composition is selected from the group consisting essentially of humulone, lupulone, xanthohumol, and iso derivatives and mixtures thereof.
 5. A process as claimed in claim 4, in which said active ingredient is lupulone.
 6. A process as claimed in claim 4, in which said active ingredient is isohumulone.
 7. A process as claimed in claim 4, in which said active ingredient is tetrahydroisohumulone.
 8. A process as claimed in claim 4, in which said active ingredient is xanthohumol. 